Controlled Activation of Non-LTR Retrotransposons in Mammals

ABSTRACT

The invention relates to nucleic acids, vector constructs which allow the controlled activation and inhibition of retrotransposition of non-LTR retrotransposons. The methods of this invention are useful for preparing said nucleic acids and vector constructs and introducing them into cells.

TECHNICAL FIELD

The invention relates to nucleic acids, vector constructs which allowthe controlled activation and inhibition of retrotransposition ofnon-LTR retrotransposons. The methods of this invention are useful forpreparing said nucleic acids and vector constructs and introducing theminto cells.

BACKGROUND ART

Transposable elements are ubiquitous in higher eukaryotic genomes.Approximately 45% of the human genome are covered by transposableelements (Lander, E. S. et al. (2001) Nature, 409, 860-921).Transposable elements are DNA sequences that can be mobilized and spreadto different positions within the genome of a single cell, a processcalled transposition. In the process, they can cause mutations andchange the amount of DNA in the genome. Transposable elements are mobilegenetic elements which are also called “jumping genes”. There is avariety of mobile genetic elements, and they can be grouped based ontheir mechanism of mobilization. Class I mobile genetic elements, orretrotransposons, are transcribed into RNA, and then reverse transcribedand reintegrated into the genome, thereby duplicating the element (“copy& paste” mechanism). The major classes of retrotransposons eithercontain long terminal repeats at both ends (LTR retrotransposons) orlack LTRs and possess a polyadenylate sequence at their 3′ termini(non-LTR retrotransposons). Class II mobile genetic elements are termedDNA transposons and are generally excised from one genomic site andintegrated into another by a “cut & paste” mechanism.

The majority of transposable elements in mammalian genomes areretrotransposons, which are considered to transpose via an RNAintermediate. Of these, the largest group comprises non-LTRretrotransposons which cover ˜42% of the human genome (Lander et al.2001, Nature 409, 880ff). Long interspersed elements (LINES) are a majorclass of non-LTR retrotransposons and cover approximately 21% of thegenome. LINE-1 retrotransposons (L1) cover about 17% of the human genome(Lander et al., 2001, Nature 409, 860-921) and play a significant rolein shaping the mammalian genome, not only through their own expansionbut also through the mobilization of non-L1 sequences. While the averagehaploid human genome harbors ˜516,000 μl copies, the subgroup of activeL1s is fairly small, encompassing 80-100 elements (Sassaman et al.,1997, Nat. Genet. 16: 37-43; Brouha et al., 2003, PNAS 100: 5280-5285).So far, 82 retrotransposition-competent, full-length L1 elements wereisolated and characterized (Sassaman et al., 1997, Nat. Genet. 16:37-43; Brouha et al., 2003, PNAS 100: 5280-5285, Moran et al. 1996, Cell87: 917-927; Kimberland et al. 1999, Hum. Mol. Genet. 8:1557-1560;Brouha et al. 2002; Am. J. Hum. Genet. 71: 327-336). L1s affected thegenome by (i) insertion of truncated L1s into new sites, (ii)intrachromosomal homologous recombination between L1s, (iii)transduction of 3′-flanking sequences during retrotransposition, (iv)aiding trans generation of processed pseudogenes and retrotranspositionof Alu elements, and (v) by causing genome instability throughsubstantial deletions (Gilbert et al. 2002, Cell 110: 315-325; Symer etal. 2002, Cell 110: 327-338; Babushok & Kazazian, 2007, Human Mutation28: 527-539).

A retrotransposition-competent, functional L1 element (RC-L1, FIG. 1)covers ˜6.1 kb and contains a 5′ untranslated region (5′ UTR) with aninternal and endogenous, CpG-rich promoter, a 1 kb ORF1 encoding aprotein (p40) of ˜40 kD with RNA-binding capability, followed by a 3.8kb ORF2 coding for a protein (p150) with a predicted molecular weight ofca 150 kD with endonuclease (EN) and reverse transcriptase (RT)activities and a cysteine-histidine-rich domain. The 3′-end of L1 isterminated by a short 3′ UTR, and a poly(A) tail (Ostertag & Kazazian,2001, Annu. Rev, Genet. 35:501-538) (FIG. 1A). L1 mRNAs are atypical ofmammalian RNAs because they are bicistronic and the mechanism oftranslation of L1 is not understood. The two ORFs are in frame andseparated by a 63-bp noncoding spacer region. Mutational analysesdemonstrated that both ORF1- and ORF2-encoded functional proteins arerequired for retrotransposition (Moran et al. 1996, Cell 87: 917-927;Feng et al. 1996, Cell 87: 905-916). At least three functions of theORF2-encoded protein were shown to be essential for retrotransposition,RT activity, EN activity and a function associated with thecysteine-histidine-rich motif. Insertion of a new L1 copy into the loosegenomic target sequence 5′-TTTT/A-3′ (Gilbert et al. 2002, Cell110:315-325; Feng et al. 1996, Cell 87: 905-916; Jurka, 1997, PNAS 94:1872-1877; Cost & Boeke, 1998, Biochemistry 37:18081-18093) is initiatedby a process termed target-primed reverse transcription (Luan et al.1993, Cell, 72: 595-605; Cost et al. 2002, EMBO J. 21: 5899-5910). Thestructure of the target site duplications (TSDs) flanking de novo L1integrants suggests a model for second strand synthesis of L1 termed“microhomology-driven single strand annealing” (Symer et al. 2002, Cell110: 327-3389; Martin & Bushman, 2001, Mol. Cell. Biol. 21: 467-475).

While the majority of L1 and other L1-mediated insertions land inintergenic and intronic sequences with little or no consequence fortheir host, occasional insertions have disrupted gene expression andcaused genetic disorders and cancer (for review Babushok & Kazazian,2007; Human Mutation 28:527-539; Ostertag & Kazazian, 2006.Retrotransposition and Human Disorders. In: Encyclopedia of LifeSciences: John Wiley & Sons. Ltd: Chichester http://www.els.net/[doi:10.1038/npg.els.0005492]. Of 53 known disease-causing insertions, 17were caused by L1 itself, while L1-mediated integrations of Alu and SVAelements caused another 33 cases; three additional cases were caused byL1-mediated insertions of simple polyA repeats. For example, germ lineL1 insertions into the factor VIII and dystrophin gene gave rise tohemophilia A and muscular dystrophy, respectively (Kazazian et al.,1988, Nature 332:164-166; Narita et al., 1993, J. Clinical Invest.91:1862-1867; Holmes et al., 1994, Nature Genetics 7:143-148). 16somatic L1-mediated retrotransposition events caused a variety ofcancers, including ALL1 rearrangement leukemias and BRCA1-associatedfamilial breast cancer (Deininger & Batzer, 1999, Mol. Genet. Metab. 67:183-193). Somatic L1 insertions into the c-myc and APC tumor suppressorgene were shown to be involved in breast and colon cancer, respectively(Morse et al., Nature 333:87-90; Miki et al., 1992, Cancer Research52:643-645). Thus, L1 is a potential mutagen and L1 retrotranspositionis mutagenic.

However, the controlled application of LINE-1 as a tool for randommutagenesis was hampered by the lack of an inducible system which allowstemporally defined, quantitative and reversible regulation of high levelLINE-1 retrotransposition in mammalian cells.

So far, only constitutive retrotransposition of marked LINE-1 reportercassettes was achieved in mammalian cell lines and in germ cells orentire organism of transgenic animals (An et al., 2006, Proc. Natl.Acad. Sci., 103: 18662-7; Ostertag, 2002, Nat. Genet. 32: 655-60; Praket al., 2003, Proc. Natl. Acad. Sci., 100: 1832-7; Babushok et al.,2006, Genome Ress., 16: 240-50). Also, LINE-1 mediated gene transfer wasonly performed with vectors expressing L1 constitutively (Kubo et al.,2006, Proc. Nad. Acad. Sci., 103: 8036-41; Soifer et al., 2001, Hum.Gene Ther., 12: 1417-28).

WO 88/03169 is merely concerned with constructs useful in yeast anddescribes a method for inducing retotransposition of yeast Tyretrotransposons in yeast using the inducible GAL1 promoter which is notfunctional in mammalian cells. Ty elements are LTR retrotansposons thatare functional in yeast whereas LINE-1s are non-LTR transposons whichare functional in mammalian cells. Consequently, the disclosedtechniques can not be transferred to mammalian cells.

Other methods relating to the constitutive expression of LINE-1s areknown in the prior art. For example, US 2003/0121063 relates to a methodfor generating a mutation in the offspring of an animal. To achieve thisgoal, mice were transfected with different non-inducible LINE1constructs and the offspring of these transgenic animals was analysed.

In addition, US 2006/0183226 discloses a new method the target-specificintroduction of certain LINE-like retrotransposons (TRAS and SMARTfamily members) into mammals. The use of inducible promoters is notdescribed. The patent application US 2006/0183226 covers a similar fieldand describes several genetic modifications of LINE-1 to achievesequence specific targeting with the modified construct.

The objective problem is to provide a vector construct and method whichallows inducible, tightly controlled and conditional expression offunctional tagged non-LTR retrotransposons, in particular, LINE-1retrotransposons in mammals. The solution of this problem is theprovision of a nucleic acid with a tetracycline-response elementoperably linked to a promoter which in turn is operably linked to atagged LINE-1 element.

SUMMARY OF THE INVENTION

The present invention relates to a nucleic acid comprising in 5′ to 3′direction (a) an inducible promoter operably linked with (b) a non-LTRretrotransposon, wherein the endogenous promoter of the non-LTRretrotransposon is genetically modified or removed to be inactive.Preferentially the inducible promoter comprises a tetracycline-responseelement (TRE) operably linked with (b) a promoter under control of thetetracycline-response element.

Another embodiment of the invention is a vector construct comprising theabove-mentioned nucleic acid.

This vector may be a viral vector or a plasmid.

A method for preparing the above-mentioned nucleic acid or theabove-mentioned vector construct are also within the scope of theinvention.

In particular, the method for transfecting cells comprises the steps ofproviding a cell comprising a promoter and a nucleic acid sequencecoding for a reversible Tet-transactivator-protein (rtTA) and expressingsaid rtTA, transfecting the cell with the vector construct, addingdoxycyline and a selection agent to the cells, selecting for cellscomprising the second marker, replacing the doxycyline and the selectionagent with a selection agent selecting for cells comprising the firstmarker, and finally obtaining the selected cells.

The invention also relates to the use of the claimed vector construct toperform insertional mutagenesis or gene trapping in human cells,isolated mammalian cells, and non-human mammals.

It is also contemplated to use the claimed vector as a medicament, inparticularly a gene-therapeutic medicament. The vector constructs of theinvention may also be used for the preparation of a medicament for thetreatment of cancer, metabolic diseases, cardiac diseases, or geneticdisorders.

DESCRIPTION OF FIGURES

FIG. 1: Structure of a functional mammalian LINE-1 retrotransposon (Fordescription see text) Abbreviations used are as follows: C,cystein-histidine-rich domain; TSD, target site duplications; UTR,untranslated region; RT, reverse transcriptase; AAAn, poly(A)tail; EN,endonuclease.

FIG. 2: A, Proof-of-Principle of inducible and tight on/off control ofL1 retrotransposition applying the Tet-On system.

In order to generate a Doxycycline (Dox)-inducible L1 retrotranspositionreporter construct using the Tet-On System, the L1.3 Retrotranspositionreporter cassette (of a mblastI -or an EGFP-tag in its 3′UTR) was setunder the control of the tetracycline-response-element (TRE) fused tothe minimal CMV promoter (P_(minCMV)).

B, Schematic of the L1 retrotransposition reporter assay: The active L1element was tagged with the indicator gene (black box in 3′UTR)containing an antisense copy of the basticidin-resistance gene(blast^(r)) disrupted by intron 2 of the gamma-globin gene in senseorientation (tsa-intron-lb) and inserted into the episomal pCEP4 vector.The splice donor (SD) and splice acceptor (SA) sites of the intron areindicated. The blast^(r) gene is also flanked by a heterologous promoter(′P) and a polyadenylation signal (′A). Transcripts originating form thecytomegalovirus (CMV) promoter drive L1 expression and can splice theintron but contain an antisense copy of the blast^(r) gene.G418-resistant (G418^(r)) colonies arise only if this transcript isreverse-transcribed, integrated into chromosomal DNA, and expressed fromits own promoter P′. Annealing sites of oligonucleotide primers GS260and Blast-B used to amplify sequences of de novo L1 integrants areindicated. Both primers are also used for “diagnostic” PCR of genomicDNA demonstrating de-novo retrotransposition: An 870-bp product isdiagnostic for a spliced out intron and shows that retrotransposition ofthe tagged L1 reporter into the genome has occurred. In contrast, a1770-bp product would come from the unspliced reporter gene cassette.

C, Experimental approach demonstrating that L1 retrotransposition occursonly after induction of L1 transcription by Dox; pTet07CMV/L1.3blas wastransfected into 2×10⁵ HeLa-M2 cells/well expressing rtTA constitutively(Hampf and Gossen 2007, J. Mol. Biol. 365: 911-920) and Doxycyclin (Dox,100 ng/ml) was added to wells 4, 5, 6, 10, 11, and 12 24 hours posttransfection, HeLa-M2 cells were selected for the presence of the L1.3retrotransposition reporter plasmid by adding 300 μg/ml Hygromycin (Hyg)to the medium in each well. Change of medium containing Hyg and Doxoccurred every 48 hours. Wells 1-6 (Plate 1): Three days aftertransfection, Hyg and Dox were removed from the medium and transfectedcells were selected for blasticidine-resistance (blast^(R), 3 μgblasticidin/ml) to select for L1 retrotransposition events. Wells 7-12(Plate 2): Five days after transfection, Hyg and Dox were removed fromthe medium and transfected cells were selected for blast^(R).

In the absence of Dox, expression of the L1 reporter was not detectablein pTet07CMV/L1.3blas-transfected HeLa-M2 cells (Wells 1-3 and 7-9),which is indicated by the appearance of only sporadic blast^(R)colonies. In contrast, the presence of Dox caused a massive activationof L1 retrotransposition events which is indicated by the growth of ˜250blase HeLa colonies per well (wells 4-6) in the case of three days ofDox induction, and ˜550 blase HeLa colonies per well (wells 10-12) inthe case of five days of Dox induction.

Abbreviations used are as follows: C, cystein-histidine-rich domain;TSD, target site duplications; UTR, untranslated region; RT, reversetranscriptase; AAAn, poly(A)tail; EN, endonuclease.

FIG. 3: A, Circular map of the episomal L1 retrotransposition reporterconstruct harbouring the inducible L1-3 element. The generation of the674-bp probe specific for the Blast-gene is depicted.

B, Northern blot analysis of total RNA extracted fromptet07CMV/L1.3blas-transfected M2-HeLa cells. Cells were harvestedbefore the addition of Dox (timepoint 0) and 6, 12 and 24 hours afterDox addition. The membrane was hybridized consecutively withL1-blast-specific and actin-gene-specific radiolabelled DNA probes.

DETAILED DESCRIPTION Definitions

The terms used herein are well known in the art and have the meaningcommonly known to the person skilled in the insofar as they are notdefined hereafter:

The term “non-LTR retrotransposons” covers four sub-types, longinterspersed nuclear elements (LINE-1s), short interspersed nuclearelements (SINEs), SVA elements (composite retrotranspsoson: SINE, VNTR,Alu), and LINE-like retrotransposons.

The term “autonomous non-LTR retrotransposons” relates to LINEs (Longinterspersed nuclear elements) (Ostertag et al., “Biology of mammalianL1 retrotransposons”, Annual Review of Genetics, 2001, vol. 35, 2001,pages 501-538.).

By “LINE1s” or “L1s” (Long interspersed nuclear elements) long DNAsequences (>5 kb, (King, Robert C. and William D. Stansfield (1997); ADictionary of Genetics. Fifth Edition. Oxford University Press.)) aremeant that represent reverse-transcribed RNA molecules originallytranscribed by RNA polymerase II into mRNA (messenger RNA to betranslated into protein on ribosomes) in mammals. LINE-1 elements codefor 2 proteins; one that has the ability to bind single stranded RNA,and another that has known reverse transcriptase and endonucleaseactivity, enabling them to copy both themselves and noncoding SINEs suchas Alu elements (see below for more detail). Generally, a functionalLINE-1 contains a 5′UTR (untranslated region) 2 ORFs (open readingframes) and a 3′UTR. In some embodiments of the invention the 5′UTR isnot included in the nucleic acid or vector construct of the invention.The 5′UTR contains an internal polymerase II promoter sequence, whilethe 3′UTR contains a polyadenylation signal (AATAAA) and a poly-A tail(Deininger P L, Batzer M A. Mammalian retroelements. Genome Research.2002; 12(10):1455-1465.). LINE1s are of mammalian origin. LINE1s in thepreferred embodiments of the invention are of human, murine or ratorigin. The nucleotide sequence for rat LINE1 is deposited at the NCBInucleotide database (GenBank accession numbers: DQ100473-DQ100482).

By “retrotransposition” as used herein, is meant the process oftranscription of a DNA sequence, reverse transcription of the resultingRNA sequence into a DNA by the LINE-1-encoded protein machinery andintegration of the DNA into a genomic site.

By a “vector construct” as used herein, a sequence of DNA is referred tothat will be propagated in eukaryotic or prokaryotic cell culture.Typical vectors are viral vectors or plasmids.

By “gene” as used herein, is meant an actual gene including both theexons and introns of the gene.

By “cDNA” as used herein, is meant a portion of a gene including onlythe exons of the gene.

By “selectable marker gene” as used herein is meant a gene or otherexpression cassette which encodes a protein which facilitates selectionof cells into which the selectable marker gene is inserted or notinserted. Examples are genes coding for blasticidin-resistance,neomycin-resistance and others.

Selection can occur on the level of two events, first, the selection forsuccessful transfection, second, the selection for successfulretransposition. For selection of transfection a selectable marker genecan be placed outside the non-LTR retrotransposon cassette into thenucleic acid or vector including a promoter functionally linked to theselectable marker gene which is active in the cell that is to betransfected. After transfection/infection said selectable substance isadded to the cell culture and only those cells will survive thetreatment that have received at least one copy of said nucleic/acidvector. The second selectable marker having its own promoter can beplaced into the 3′ UTR of the non-LTR retrotransposon or any other partof the nucleic acid coding for the non-LTR retrotransposon as long asthere is no interference with the retrotransposition event. Theselectable marker and the promoter controlling its transcription may bein reverse direction to the promoter controlling the transcription ofthe non-LTR retrotransposon. To avoid transcription of the secondselectable marker gene before retrotransposition the second selectablemarker gene is interrupted by an intron, e.g. a small artificialgamma-globin intron or another intron. Thus transcription of theselectable marker gene before retrotransposition will result in anon-functional protein. The intron is constructed in a way thattranscription of the cassette will lead to removal of the intron. Thesplice acceptor and donor sites are placed accordingly and as known inthe state of the art. Only after reintegration of said transcribedcassette into the genome of the host the selectable marker gene can beexpressed indicating the successful retrotransposition event.

By “reporter gene” as used herein is meant a gene or other expressioncassette which encodes a protein which facilitates identification ofcells into which the reporter gene is inserted. Examples are genescoding for ‘Green Fluorescent Protein (GFP), Enhanced GFP (EGFP),β-Galactose.

By “host” is meant any biological system that supportsretrotransposition events in the genome of the host. Such a biologicalsystem may be an animal, a mammalian animal, particularly a human.Cells, cell-lines or tissues derived from said animals are also regardedto be hosts.

By “therapeutic gene” as used herein is meant a gene or other expressioncassette which encodes a protein or nucleic acid whoseexpression/transcription results in a therapeutic effect. The expressionof the therapeutic gene may complement a gene that is not expressed inthe host or in reduced amounts wherein the reduced or lacking expressionof said host gene causes a disease or a disorder in the host. Thetherapeutic gene may also be a dominant negative mutant whose expressionreduces or eliminates the effects of the expression of a host gene thatcauses a disease or genetic disorder in the host.

By “heterologous DNA” as used herein, is meant DNA which is notnaturally found in the cell into which it is inserted or the nucleicacid into which it is inserted. For example, when mouse or bacterial DNAis inserted into the genome of a human cell, such DNA is referred toherein as “heterologous DNA”. If a bacterial gene is inserted intomammalian or human retrotransposon, such DNA is referred to herein as“heterologous DNA”. If a mammalian or human gene is inserted intomammalian or human retrotransposon and said mammalian or human gene iscommonly not present in said retrotransposon, such DNA is referred toherein as “heterologous DNA”. In contrast, the term “homologous DNA” asused herein, denotes DNA which is found naturally in the cell or nucleicacid into which it is inserted. For example, the insertion of mouse DNAinto the genome of a mouse cell constitutes insertion of “homologousDNA” into that cell. In the latter case, it is not necessary that thehomologous DNA be inserted into a site in the cell genome in which it isnaturally found; rather, homologous DNA may be inserted at sites otherthan where it is naturally found, thereby creating a genetic alteration(a mutation) in the inserted site.

By “non-L1 DNA” as used herein, is meant DNA which does not naturallyoccur in an L1 element.

It will be appreciated that the invention should not be construed to belimited in any way to the precise DNA sequences which are disclosedherein. Homologous DNA sequences having substantially the same functionas the disclosed DNA sequences are also considered to be included in theinvention.

As used herein, the term “homology” refers to the subunit sequenceidentity or similarity between two polymeric molecules e.g., between twonucleic acid molecules, e.g., between two DNA molecules, or twopolypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit, e.g., if a positionin each of two polypeptide molecules is occupied by phenylalanine, thenthey are identical at that position. The homology between two sequences,most clearly defined as the % identity, is a direct function of thenumber of identical positions, e.g., if half (e.g., 5 positions in apolymer 10 subunits in length) of the positions in two polypeptidesequences are identical then the two sequences are 50% identical; if 70%of the positions, e.g., 8 out of 10, are matched or homologous, the twosequences share 80% identity. By way of example, the polypeptidesequences MCDEFG and MCDHIK share 50% identity and the nucleotidesequences GAATCG and GAAGAC share 50% identity. Nucleotide sequenceidentity can be determined using a known computer program. For example,amino acid or nucleotide sequences can be aligned by an alignmentprogram such as CLUSTAL W (Thompson, J. D. et al., 1994, Nucleic AcidsRes. 22: 4673-80), and identity can be calculated by counting thematching nucleotides. Gaps are treated in the same way as mismatches,and identity can be calculated as the ratio of matched nucleotideswithin the total number of nucleotides comprising the gaps.Alternatively, programs such as blastn can be used (Altschul, S. F. etal. (1990) J. Mol. Biol. 215: 403-410; Gish, W. & States, D. J. (1993)Nature Genet. 3: 266-272; Madden, T. L. et al. (1996) Meth. Enzymol.266: 131-141; Altschul, S. F. et al. (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J. & Madden, T. L. (1997) Genome Res. 7:649-656). Forexample, in BLAST 2 SEQUENCES, which compares two amino acid sequencesor nucleotide sequences by blastp or blastn, respectively (see TatianaA. Tatusova, Thomas L. Madden [1999], “Blast 2 sequences—a new tool forcomparing protein and nucleotide sequences”, FEMS Microbiol. Lett. 174:247-250; the NCBI website for BLAST 2 SEQUENCES[http://www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html]), BLOSUM62 is usedas the matrix for scoring when comparing amino acid sequences (Henikoff,Steven and Jorga G. Henikoff [1992] Amino acid substitution matricesfrom protein blocks. Proc. Natl. Acad. Sci. USA 89: 10915-19; Open gappenalty: 11, extension gap penalty: 1). Identity values can be obtainedas Identities (%) by searching without the use of FILTER (filtering ofLow-complexity sequences). In particular, nucleic acids having 99%, 98%,97%, 96%, 95%, 90%, 80%, 70%, or 60% identity with the nucleic acids ofthe invention are claimed.

An “isolated DNA”, as used herein, refers to a DNA sequence which hasbeen separated from the sequences which flank it in a naturallyoccurring state, e.g., a DNA fragment which has been removed from thesequences which are normally adjacent to the fragment, e.g., thesequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid (e.g., cellular components, RNA, DNA or protein) in itsnatural state. “Complementary”, as used herein, refers to the subunitsequence complementarity between two nucleic acids, e.g., two DNAmolecules. When a nucleotide position in both of the molecules isoccupied by nucleotides normally capable of base pairing with eachother, then the nucleic acids are considered to be complementary to eachother at this position. Thus, two nucleic acids are complementary toeach other when a substantial number (at least 50%) of correspondingpositions in each of the molecules are occupied by nucleotides whichnormally base pair with each other (e.g., A:T and G:C nucleotide pairs).The skilled person will also appreciate that artificial bases exist thatcan pair with natural occurring bases or with other artificial bases(e.g. phosphorothioate or PNA derived bases).

“Positioned in an antisense orientation with respect to the direction oftranscription of the DNA” as used herein, means that the transcriptionproduct of the DNA, the resulting mRNA, does not encode the polypeptideproduct specified by the “sense” strand of DNA. Rather, the mRNAcomprises a sequence which is complementary to an mRNA which encodes theprotein product.

The term “insertional mutation” is used herein to refer thetranslocation of nucleic acid from one location to another locationwhich is in the genome of an animal so that it is integrated into thegenome, thereby creating a mutation in the genome.

A “retrotransposition event” is used herein to refer to the duplicationof a retrotransposon sequence via a ‘Copy & Paste’ mechanism with thepreferable outcome being integration of a new retrotransposon copy intoa new genomic location. The genome is preferentially a mammalian orhuman genome.

The term “detecting the DNA molecule” is used herein to refer to methodswell known in the art for identifying a specific nucleic acid sequenceamongst other nucleic acid sequences, including but not limited to, PCR,RT-PCR, Southern hybridization, Northern hybridization, single strandconformation polymorphisms, and the like.

The term “cell” refers to any cell type. The cell can be part of anorganism or isolated. Isolated cells are removed from the organism.Cells can be primary cells or cell-lines. Cells may be prokaryotic,preferentially eukaryotic, yet more preferentially mammalian and/orhuman.

The term “tissue” refers to any tissue type. The tissue can be part ofan organism or isolated. Isolated tissues are removed from the organism.Tissues are preferentially from a mammalian and/or human source.

The term “promoter” relates to a specific DNA sequence that isrecognized by proteins known as transcription factors. These factorsbind to the promoter sequences, recruiting RNA polymerase, the enzymethat synthesizes the RNA from the coding region of the gene. In this waya gene is transcribed. Promoters can be active or inactive. In theformer case transcription can occur starting from the promoter, in thelatter case transcription of the gene does not occur.Activation/inactivation can occur if the promoter is under the controlof operator sequence and certain factors are bound or not bound to theoperator sequence activating/deactivating the promoter. An example forsuch a control mechanism is the ‘Tet-System’ explained below.Alternatively a promoter can be inactivated by genetically modifying thepromoter, i.e. the nucleic acid sequence of a promoter that is usuallyconstitutively active is altered in a way that transcription can nolonger be initiated at said promoter. The promoter may be an exogenouspromoter, i.e., a promoter that is not naturally found in non-LTRretrotransposons.

“Genetically modifying” relates to the replacement, addition, ordeletion of nucleic acids in a nucleic acid sequence.

The term “codon optimized” or “codon optimized gene” relates to a genein which the codons in a nucleic acid sequence of an open reading frameare altered to codons which are the most favored codons in highlyexpressed mammalian genes. However, the codons are altered withoutallowing a change of the encoded amino sequences. These altered “codonoptimized” genes are especially useful for non-LTR retrotransposons,since in this way the transcriptional elongation defect which isreported to participate in the control of wildtype L1 retrotranspositionfrequency is bypassed (Feng et al. 1996, Cell, 87: 905-916; Moran et al.1996, Cell: 917-927; Cost et al. 2002, EMBO J., 21: 5899-5910; Han andBoeke, 2004, Nature, 429: 314-318). As a consequence of thecondon-optimization the transposition level of a non-LTRretrotransposon, in particular, a L1 retrotransponson or a geneticallymodified version of a L1 retrotransposon may increase by about 200-fold(Han and Boeke, 2004, Nature, 429: 314-318; An et al. 2006, PNAS, 103:18662-7).

Nucleic Acids of the Invention

The nucleic acid of the invention may comprise, in 5′ to 3′ direction,an inducible promoter being operably linked with a non-LTRretrotransposon.

The non-LTR retrotranspon may be an autonomous non-LTR retrotranspon.

In a preferred embodiment, the nucleic acid of the invention maycomprise in 5′ to 3′ direction a tetracycline-response element beingoperably linked with a promoter under control of thetetracycline-response element, and being operably linked with a non-LTRretrotransposon.

The promoter may be any promoter that can be controlled by atetracycline-response element, in particular, the immediate-earlypromoter of cytomegalovirus. The tetracycline-response element may alsobe operably linked with the constitutive composite chicken β-actinpromoter CAG (Lobe et al. 1999, Dev. Biol. 208: 281-292); the chickenβ-actin promoter combined with the CMV immediate early enhancer(Hakamata et a1.2001, BBRC 329: 288-295); the Ubiquitin-C promotercombined with CMV enhancer (Lois et al. 2002, Science 295: 868-872); theRNA-Polymerase II promoter; viral promoters, in particular theespecially SV 40 promoter; house keeping promoter, in particular theactin-, PGK-, DNA polymerase II- and ubiquitin promoter; germline-specific promoters, in particular, the mouse heat-shock protein70-2 promoter (pHsp70-2), which is known to produce strong transgeneexpression in meiosis of male spermatogenesis (Dix et al. 1996, Dev.Biol. 174:310-321); the neuron-specific GnRH (gonadotropin-releasinghormone)-promoter.

In an alternative embodiment, the non-LTR retrotransposon is operablylinked with an optimized ecdysone responsive promoter (No et al. 1996;Proc. Natl. Acad. Sci. USA 93: 3346-3351). The ecdyson-inducibleexpression system utilizes a heterodimer of the ecdyson receptor (VgEcR)and the retinoid X receptor that binds a hybrid ecdysone responseelement in the presence of the synthetic analog of ecdysone muristeroneA (No et al. 1996; Proc. Natl. Acad. Sci. USA 93: 3346-3351). Binding ofthe heterodimer to the modified ecdysone response element in the minimalpromoter, activates L1 transcription.

The non-LTR retrotransposon may further comprise a heterologous geneselected from the group consisting of a reporter gene, a therapeuticgene, and a selectable marker gene.

The reporter gene may be selected from the group consisting of GFP,β-Galactose.

An antisense GFP construct may be generated which replaces the neo^(R)marker gene in the 3′-UTR of L1. As before, the initiation codon ATG ofthe GFP gene is replaced and a splice acceptor sequence is placed at the5′ end of the GFP gene. GFP expression will only occur if the geneproduct is expressed as an in-frame fusion protein. The N-terminus ofthis fusion protein is derived from the host gene into which the DNAinserted. A similar strategy can be used applying lacZ or compositeindicator genes such as lacZ/neo^(R) or GFP/neo^(R). Furthermore, it ispossible to generate three independent constructs to ensure thatinsertions can be identified in all three reading frames. A furtherheterologous gene may be the herpes simplex virus (HSV) thymidine kinasegene.

The selectable marker gene is selected from the group consisting of drugresistance genes, in particular the blasticidin resistance gene(Blast^(R)=blasticidin S deaminase), the neomycin resistance gene(neo^(R)=neomycin phosphotransferase), the hygromycin resistance gene(hyg^(R)), the puromycin resistance gene (puro^(R)), the zeocinresistance gene (zeo^(R)), the hgprt gene (=hypoxanthine guaninephosphoribosyl transferase, a human gene which can be selected for inhuman cells in culture).

The therapeutic gene may be a gene or other expression cassette whichencodes a protein or nucleic acid whose expression/transcription resultsin a therapeutic effect. The expression of the therapeutic gene maycomplement a gene that is expressed in reduced amounts in the host ornot at all wherein the reduced or lacking expression of said host genecauses a disease or a disorder in the host. The therapeutic gene mayalso be a dominant negative mutant whose expression reduces oreliminates the effects of the expression of a host gene that causes adisease or genetic disorder in the host.

The heterologous gene may be under control of a promoter and operablylinked with this promoter which is different from the promotercontrolling the transcription of the non-LTR retrotransposon. Thenucleic acid sequence comprising the heterologous gene and the promoteroperably linked to it may be in inverse direction to the promotercontrolling transcription of the retrotransposon. This heterologous genemay also contain an intron. This intron may be designed to have splicedonor and splice acceptor sequences which are in the same orientation asthe transcriptional orientation of the L1 retrotransposon. The intronmay be constructed in a way that if transcription starts at theheterologous gene's promoter, the resulting messenger RNA will betranslated into a protein that is not functional. Not functional meansthat it does not have the function usually ascribed to the proteinencoded by the heterologous gene. The promoter of the heterologous geneand the promoter of the retrotransposon may be the same or different.The promoter of the heterologous gene may be inducible or not inducible.If the promoter of the heterologous gene is also inducible it would benecessary to use a promoter that is different from the TRE-P_(min) CMVpromoter cassette which controls L1 transcription. This would facilitateto induce L1 transcription and expression of the heterologous geneindependently from each other by different factors. That way it would bepossible to trigger retrotransposition by adding Doxycyclin to thesystem, without affecting expression of the heterologous gene.Expression of the heterologous gene could then be induced at a desiredlater time point. This would be very advantageous if, e.g. a therapeuticor other gene is retrotransposed with the nucleic acid of the inventioninto a host, e.g. in a time- and/or tissue-specific way. Following L1retrotransposition, expression of the heterologous, preferentially,therapeutic gene may be induced at a defined time point for a definedperiod of time. Thus, expression of a therapeutic gene or othertreatment would be under a more efficient control than if only theretrotransposition process was regulated. Unnecessary transcription ofthe heterologous gene could be avoided.

Referring to FIG. 2A, the promoter controlling the expression of theheterologous gene localized in the 3′-UTR region of L1 can be either anRNA Polymerase II—or an RNA-Polymerase III promoter. Examples of RNA PolII promoters which are useful include, but are not limited to,housekeeping promoters, such as actin, PGK, DNA Pol III or a ubiquitinpromoter; tissue-specific promoters, like albumin, globin, ovalbuminpromoter sequences, skin specific promoters such as K12 or K14,inducible promoters, like steroid inducible promoters, the L1 elementpromoter, viral promoters, like SV40 early promoter, the Rous sarcomavirus (RSV) promoter and the cytomegalovirus immediate early promoter(CMV) and other retroviral LTRs. RNA polymerase III promoters which arewithin the scope of the invention are, tRNA promoters and the 5 S RNApromoter.

Additional promoters that may be used to control expression of theheterologous gene are: constitutive composite chicken β-actin promoterCAG (Lobe et al. 1999, Dev. Biol. 208: 281-292); chicken (3-actinpromoter combined with the CMV immediate early enhancer (Hakamata eta1.2001, BBRC 329: 288-295); ubiquitin-C promoter combined with CMVenhancer (Lois et al. 2002, Science 295: 868-872); germ line-specificpromoters like mouse heat-shock protein 70-2 promoter (pHsp70-2) knownto produce strong transgene expression in meiosis of malespermatogenesis (Dix et al. 1996, Dev. Biol. 174:310-321);neuron-specific GnRH (gonadotropin-releasing hormone)-promoter.

It is also appreciated that both promoters are active in the same tissueand are the same or different promoters. Even tissue specific promotersmay be leaky in the sense that retrotransposition may not occur in theintended tissue but also in other not intended tissues. The probabilityof leakage will be already reduced if the same two specific promotersare used. This would result in a more tissue specific expression of theheterologous or therapeutic gene. The specificity may even be increasedby using two promoters which are specific for the same cell type ortissue but have different sequences, i.e. are of different types.

The non-LTR retrotransposon may be the mammalian long interspersedelement 1 (LINE 1). The LINE-1 sequence may consist of a LINE-1 5′ UTR,ORF 1, ORF 2, 3′ UTR and a Poly A signal. The LINE-1 sequence mayconsist of a LINE-1 ORF 1, ORF 2, 3′ UTR and a Poly A signal. Theexperimental data provided by the present inventors indicated that theendogenous promoter of the non-LTR retrotransposon needs to beinactivated in the nucleic acid of the invention in order to tightlycontrol L1 transcription. It was surprisingly found that without suchinactivation the non-LTR retrotransposon is constitutively transcribedeven if operably linked to an inducible promoter. Thus transcriptionoccurs independent of the activation/deactivation of thetetracycline-response element in the nucleic acid. Therefore, it wasimpossible to produce an inducible system for non-LTR retrotransposonsusing the techniques disclosed in the prior art. For rendering thenon-LTR retrotransposon inducible it is therefore required to inactivatethe endogenous promoter of the non-LTR retrotransposon. Inactivation ofthe promoter can be achieved in many ways, like genetically modifyingthe promoter sequence, in particular, by replacing the promoter sequencewith any sequence that is not a promoter sequence in mammalian cells.Alternatively, the entire promoter region may be deleted. In a furtheralternative embodiment the 5′UTR of the non-LTR retrotransposon or ofthe LINE1 may be deleted. In the present invention it is essential toremove the promoter sequence, e.g., by removing the 5′ UTR, to ensurethat the regulation of L1 expression by the Tet-On system is tightlycontrolled (i.e, not leaky) and that the lack of Dox allows onlysporadic and negligible L1 expression (FIG. 2C).

In a further embodiment of the invention the long interspersed element 1(LINE-1) is selected from the group consisting of human, rat, and mouseLINE-1. In yet a further embodiment of the invention the mammalian longinterspersed element 1 (LINE-1) is codon-optimized.

Vector Constructs of the Invention

Vector constructs of the invention may comprise any of the above recitednucleic acids of the invention.

The vector construct may be selected from the group consisting of aviral vector and a plasmid.

Viral vectors are well known in the art and be derived from any knownvectors. Especially, vectors that can be used to transfer nucleic acidsinto mammalian, in particular, human cells, are appreciated.

The nucleic acids of the invention may be introduced into cells usingviral vectors with a tropism for mammalian cells. The vector sequencemay comprise DNA sequences derived from a virus, such as, but notlimited to, Epstein Barr virus (EBV) comprising oriP and EBNA1 or apolyoma-based virus comprising the polyomavirus origin of DNAreplication and a polyomavirus enhancer sequence. Other viral vectorsuseful in the invention include adenoviruss, adeno-associated virus,lentivirus, parvovirus, herpes simplex virus, retroviruses, poxviruses,and the like. These sequences comprise a eukaryotic origin of DNAreplication to facilitate replication of the DNA molecule in aeukaryotic cell. Note, however, that certain vectors, such asadeno-associated virus, may be replication deficient, but may be stilluseful because they provide efficient delivery vehicles for introductionof the DNA into the desired target cell. It is not necessary that thevector sequences be limited to naturally occurring eukaryotic viralelements, e.g., mammalian artificial chromosomes are also contemplatedin the invention.

The vector construct may contain a nucleic acid coding for a furtherselectable marker outside of the non-LTR retrotransposon, wherein saidheterologous gene of the non-LTR retrotransposon will only betranscribed after the non-LTR retrotransposon has integrated into thegenome of a cell.

The above-mentioned vectors may be constructed using known vectorsystems. By using viral vectors, the invented L1 expression cassette canbe introduced efficiently into host cells, and RNAs and ORF-encodedproteins can be expressed at high levels. Those viral vectors that donot integrate into chromosomes are especially preferable. By using thistype of vector, components necessary for retrotransposition can betransiently expressed in target cells. Since these vectors will beremoved from cells over time, they will not be unnecessarily expressedafter retrotransposition is complete, and are therefore vectors veryuseful in the present invention. Examples of viral vectors that do notintegrate into chromosomes include adenovirus vectors (for example,pShuttle, Clontech), Sendai virus vectors, vaccinia virus vectors,Epstein-Barr virus vectors, baculovirus vectors, herpes virus vectors,and sindbis virus vectors (Soifer, H. et al., 2001, Hum. Gene Ther. 12:1417-1428; Kay, M. et al., 2001, Nat. Med. 7: 33-40; Kubo et al. 2006,PNAS 103: 8036-41). By using a vector that integrates into chromosomes,the integration site may be regulated. Examples of vectors thatintegrate into chromosomes include retrovirus vectors, lentivirusvectors, adeno-associated virus vectors, and foamy virus vectors. Theseviral vectors can be prepared by methods well known to those skilled inthe art. Viral vectors can be purified, for example, by centrifugation,according to their types.

In order to express the vectors of the present invention in animals, invivo or ex vivo, DNA vectors such as plasmids can be administeredtogether with transfection reagents such as cationic lipids orliposomes, e.g. Gene Juice (Novagen), FuGene (Roche). Naked DNAs orviral vectors can be directly administered. Examples for administrationtargets are humans and non-human mammals, and administration can beperformed ex vivo or in vivo, to cells, tissues, organs, and such.Administration to a living body may be performed ex vivo or in vivo. Inthe case of in vivo applications, the vector of the present invention isadministered directly to a living body. In the case of ex vivoapplications, administration to cells outside a living body is followedby the introduction of those cells into a living body. As an example foran ex vivo method, cells producing a viral vector of the presentinvention may be administered.

Vector constructs in the sense of the invention is a nucleic acidincluding a vector as described above and an inducible non-LTRretrotransposon.

When administering locally to a target tissue, vector constructs orcells are administered to the target tissue via an injection needle,catheter, or such. Alternatively, vector constructs can be introduced totarget tissues using carriers that can deliver vector constructs tospecific tissues.

Thus, the vector constructs of the present invention can specificallyfacilitate L1 retrotransposition in tumor cells and such.

The vector constructs of the present invention can be mixed with knowncarriers and vehicles to form composites. The vector constructs of thepresent invention can also be administered as pharmaceuticalcompositions that are formulated by conventional preparation methods.For example, they can be prepared as compositions by mixing them withpharmaceutically acceptable carriers or vehicles, which specificallyinclude sterilized water or physiological saline, salts, vegetable oil,stabilizers, preservatives, suspensions, and emulsifiers. Furthermore,the vector construct of the present invention can be prepared ascompositions for introducing nucleic acids into cells together withliposomes or cationic lipids.

When administered as pharmaceutical agents to a living body, the vectorconstructs of the present invention can generally be administeredlocally or systemically by methods well known to those skilled in theart, such as intraarterial injection, intravenous injection,subcutaneous injection, and intramuscular injection. Alternatively, theycan be administered locally through a syringe, catheter, needle-lessinjector, or such. Dosage can vary depending on a patient's weight andage, the method of administration, and the symptoms, but one skilled inthe art can appropriately select an appropriate dose. Administration canbe performed once, or a number of times. Administration of the vectorconstructs of the present invention can be performed according toconventional gene therapy protocols.

Methods of the Invention

A method of the invention relates to providing a cell comprising anucleic acid of the invention as described above and inducing thetranscription of the non-LTR. In a preferred embodiment, the method ofthe invention for transfecting cell culture cells comprises the steps ofproviding a cell comprising a nucleic acid comprising a promoter and anucleic acid sequence coding for a tetracycline-controlledtransactivator protein (tTA) and expressing said tTA or coding for areverse tetracycline-controlled transactivator protein (rtTA) andexpressing said rtTA, then transfecting the cell with the vectorconstruct described above and adding doxycyline and a selection agent tothe cells, wherein the selection agent positively selects cellsexpressing the selectable marker gene outside of the marked LINE-1retrotransposon. Then a selection for cells comprising said selectablemarker gene can be performed which is followed by replacing thedoxycycline and the selection agent with another selection agent,wherein the selection agent positively selects cells expressing theheterologous marker gene localized in the 3′-UTR of the LINE-1retrotransposon. Following this step the cells comprising theheterologous marker gene are selected. Finally, selected cells areobtained of at least one de-novo L1-retrotransposition event. In analternative embodiment, the heterologous marker gene may be replaced orsupplemented by a reporter gene, e.g., GFP. In that case, identificationand possible selection of retrotransposed cells can occure visually,e.g., by inspection of cells in culture or inspection of tissues samplesfrom animals.

In another embodiment expression of the reverse tertracycline-controlledtransactivator-protein (rtTA) is replaced with the expression of thetetracycline-controlled transactivator-protein (tTA). In that caseadding doxycycline is shutting down expression of theTRE-P_(minCMV)-controlled L1-retrotransposition reporter cassette (FIG.1).

Further details of the application of the ‘Tet-On’ system in transgenicmice are summarized in Schonig & Bujard, “Generating conditional mousemutants via Tetracycline-controlled gene expression”, Methods inMolecular Biology, vol 209: pp 69-104, 2003 and in Sprengel & Hasan,“Tetracycline-controlled genetic switches”, HEP, vol 178: 49-72, 2007,and c in Kistner et al., “Doxycyclin-mediated quantitative andtissue-specific control of the gene expression in transgenic mice”,Proc. Natl. Acad. Sci. USA, vol. 93, pp. 10933-10938, 1996.

In a further embodiment the nucleic acid sequences coding for rtTA ortTA are functionally linked to a tissue-specific or ubiquitous,constitutively active promoter. In this way, the invention provides thebenefit of providing a non-LTR retrotransposon that is not onlyinducible but will be induced only in desired tissues or defined celltypes of the animal organism. This characteristic of the invention isvery useful in transgenic animals when the expression of a nucleic acidis intended to be restricted to a particular pathologic tissue or celltype affected by disease. No expression would occur in healthy tissuesand cell types of an animal. Thereby, undesirable side effects may bedramatically reduced. Alternatively, the promoter may be constitutive.For preparing such transgenic animals, an animal comprising the nucleicacid of the invention may be bred with an animal comprising a nucleicacid coding for rTA or rtTA, respectively.

In another embodiment the nucleic acid comprising a promoter and thenucleic acid sequence coding for the rtTA is provided by transfectingthe cells with a vector containing said nucleic acid.

In yet another embodiment the nucleic acid comprising a promoter and thenucleic acid sequence coding for the tTA is provided by transfecting thecells with a vector containing said nucleic acid.

In another embodiment a cell is provided and the cell is co-transfectedwith the nucleic acid comprising a promoter and the nucleic acidsequence coding for the rtTA and one of the above defined vectorconstructs encoding the Tet/Dox-inducible L1 reporter cassette.

In yet another embodiment a cell is provided and the cell isco-transfected with the nucleic acid comprising a promoter and thenucleic acid sequence coding for the tTA and one of the above definedvector constructs encoding the Tet/Dox-inducible L1 reporter cassette.

The Tet System

The Tet system exists in the form of the Tet-Off and the Tet-On System:

In the Tet-Off System the tetracycline-response element (TRE) is locatedupstream of the minimal immediate early promoter of cytomegalovirus(P_(minCMV)), which is silent in the absence of activation. tTA(Tetracycline-controlled transactivator) binds the TRE—and therebyactivates transcription of the LINE-1 retrotransposon—in the absence ofDox;

In the Tet-Off system, tTA binds the TRE and activates LINE-1transcription in the absence of tetracycline or Dox.

In the Tet-On System the ‘reverse’ Tet repressor (rTetR) was created byfour amino acid changes of the Tet repressor that reverse the protein'sresponse to Dox. As a result of these changes, the rTetR domain of thereversible Tet-transactivator (rtTA=fusion of rTetR and the C-terminal127 amino acids of the Herpes simplex virus VP16 activation domain)binds the TRE and activates transcription of the L1 reporter cassette inthe presence of Dox. In a preferred embodiment, the gene of interest isthe L1 retrotransposition reporter cassette.

In the Tet-On system, rtTA binds the TRE and activates transcription ofLINE1 in the presence of Dox (see also Clontech, Tet systems usermanual, 2007).

In a further embodiment of the invention, the nucleic acid whichcontains an inducible promoter pursuant to the Tet-On system is combinedwith a tetracycline-controlled transcriptional silencer. If the Tet-Onsystem is used for induction of the tetracycline-controlled, conditionalLINE-1 (L1) retrotransposition in mammalian cells, the system can becombined with a tetracycline-controlled transcriptional silencer (tTS)to minimize the background expression in absence of doxycycline (Dox).tTS is a fusion protein of the tet repressor (TetR) and the KRAB-ABsilencing domain of the Kid-1 protein, a powerful transcriptionalrepressor (Freundlieb S. et al. 1999, J. Gene Med. 1: 4-12; Witzgall R.et al. 1994, Proc. Natl. Acad. Sci USA 91: 4514-4518). In the absence ofDox, tTS binds to the tetO7 sequence in the tet responsive element (TRE)of the otetO7CMV/L1.3blas and blocks expression and thusretrotransposition of the L1 element. In the presence of Dox, the tTSdissociates from the TRE, allowing the Dox dependent binding of rtTA tothe TRE and activating the expression of L1.

Alternative Systems with Inducible Promoters

In an alternative embodiment, the non-LTR retrotransposon is operablylinked with an optimized ecdysone responsive promoter (No et al. 1996;Proc. Natl. Acad. Sci. USA 93: 3346-3351). The ecdyson-inducibleexpression system utilizes a heterodimer of the ecdyson receptor (VgEcR)and the retinoid X receptor that binds a hybrid ecdysone responseelement in the presence of the synthetic analog of ecdysone muristeroneA (No et al. 1996; Proc. Natl. Acad. Sci. USA 93: 3346-3351). Binding ofthe heterodimer to the modified ecdysone response element in the minimalpromoter, activates L1 transcription.

Targeted Integration of Tet/Dox-Inducible Marked L1 Elements

Mammalian wildtype LINE-1s integrate randomly into the consensusinsertion sequence 5′-TTTT/A-3′ into the host genome. To achieve sitespecific insertion of DNA into the host genome, a gene coding for asequence-specific or protein-specific DNA-binding domain may bepositioned somewhere within the L1-coding sequence. The specific domainmay include a p53 binding domain, a zinc finger binding domain, type IIendonuclease binding domain, a homeobox binding domain, APE-typeendonucleases, and other domains. In this way, the mammalian LINE-1retrotransposons can be directed to areas next to genes that arecontrolled by the above-mentioned factors or to genomic regions wherede-novo L1 integration is not harmful to the host cell (cf.US2006/0183226, page 1 et seq.).

Isolation of Host-Encoded Genes Flanking the De-Novo L1 Insertion Site

In order to facilitate the isolation of host genes flanking theinsertion site the L1 cassette may be modified in a way so that itcontains an origin of replication (ori), e.g. prokaryotic on oreukaryotic on (yeast oris, like 2 micron, or ARS/CEN and others) in the3′ UTR region or between the start of the 3′ UTR and the polyA signal.Independent of such modification, after a successful de-novo L1retrotransposition event the host-genome DNA can be digested with arestriction enzyme cleaving outside of the cassette or a region thereof.Subsequently the isolated host genome DNA may be religated andpropagated in prokaryotic or eukaryotic cells depending on the selectedon (cf. US2006/0183226, page 9, section 80).

Gene-Trap Technology

The claimed constructs and methods can also be combined with gene-traptechnology. For example, the promoter and initiation codon areeliminated and an intron acceptor splice site is added in place thereof.After retrotransposition the heterologous marker gene may be placed 3′to a host gene controlled by a host promoter. Thus, the heterologous(marker) gene can only be expressed as a fusion protein. Naturally, thisspecific method will only detect expressed host genes and allow thedetection of insertional mutations in or close to expressed genes.

For example, a selectable marker gene can be introduced into the 3′ UTRas described above. The promoter driving expression of said selectablemarker gene is removed and a splice acceptor signal is inserted at thestart codon of the selectable marker gene; a bacterial promoter (whichis only transcribed in bacteria) driving expression of a markerselectable in bacteria and an origin of replication, are introduceddownstream of the indicator gene. If the LINE-1 element retrotransposesinto a desired region of the genome (site specific or not sitespecific), the selectable marker gene is spliced into mRNA. If thesplicing event places the selectable marker gene in frame with thepreceding exons, the selectable marker gene mRNA is translated and cellsexpressing the marker can be selected. Three different constructs may bedesigned such that all three reading frames of marker DNA are readthereby ensuring expression of protein from any spliced mRNA. Thepresence of the bacterial promoter and origin of replication downstreamof the indicator gene should not interfere with splicing, but will allowfor the simple isolation of the retrotransposed genomic LINE-1retrotransposon insertions using methods similar to those describedherein.

This strategy can also be used for the production of specific knock-outmutants via L1 insertion.

The “marker-gene” will then be an antisense sequence of the gene to beknocked out resulting in a hairpin structure that cannot be transcribed.

Alternatively, a “promoter trap” can be constructed by providing anindication gene with an initiation codon but lacking a promoter. Theindication gene will be detectable if the cassette is integrated closeto an active promoter which can then be identified.

An “enhancer trap” can also be designed by combining the L1 cassettewith an indicator gene of an initiation codon and a weak promoter thatdoes not lead to transcription without the presence of any enhancer.

Detection of De-Novo L1 Retrotransposition Events

De-novo LINE-1 insertions retrotransposed by the methods of the presentinvention can be detected by Southern blot analysis of host chromosomalDNA, by in situ hybridization of chromosomes such as FISH, or by‘Extension Primer Tag selection’ (EPTS)-LM-PCR (Schmidt et al. 2001,Hum. Gene. Ther. 12: 743-49) or Inverse PCR (An et al. 2006, PNAS,103:18662-18667). The polymerase chain reaction (PCR) which is wellknown in the art can also be used to detect retrotransposition events(Sambrook, J et al., Molecular Cloning 2^(nd) ed., 9.47-9.58, ColdSpring Harbor Lab. press, 1989; “The PCR Technique: DNA sequencing”(Eds. J. Ellingboe and U. Gyllensten), “BioTechniques Update Series”,Eaton Publishing, 1999; “The PCR Technique: DNA sequencing II” (Eds. U.Gyllensten and J. Ellingboe), “BioTechniques Update Series”, EatonPublishing, 1999; “PCR Technology: principles and application for DNAamplification” Ed. by H. A. Erlich, 1989, Stockton Press). By usingprimer pairs for the PCR analyses that are specific for the heterologousgene positioned within the 3′ UTR, it is possible to distinguish agenomic, spliced and retrotransposed copy of the L1 reporter elementfrom an unspliced original L1 cassette of our invention (FIG. 2A).Genomic sequences flanking de-novo L1 insertions can be identified byEPTS-LM-PCR (Schmidt et a1.2001, Hum. Gene. Ther. 12: 743-49) or InversePCR (An et al. 2006, PNAS, 103:18662-18667).

ADVANTAGES OF THE INVENTION

1. In the case of transient cotransfections performed in cell cultureexperiments:

The ability to induce L1 retrotransposition for a defined period of timemakes it possible to avoid consecutive transfections in order toevaluate the effect of certain gene products on L1 retrotransposition.Series of consecutive transfections produce more stress for transfectedcells than a single transfection event and can cause secondary effectsinfluencing the outcome of an experiment. The vectors and nucleic acidsof the invention allow the cotransfection of an expression constructwhose gene product may have an impact on L1-retrotransposition with anL1 reporter construct to induce L1 expression precisely for the periodof time, the expression of the concurrently transfected expressionconstruct is detectable.

2. This invention will permit conditional expression of a functionaltagged L1 retrotransposon in double transgenic mice/rats, i.e. L1expression can reversibly be adjusted in a time-dependent anddose-dependent manner. A mouse model applying the invention allows tomonitor retrotransposition frequency in defined tissues and cell typesduring defined periods of development.

3. In the case of an ex vivo gene therapeutic approach using L1retrotransposons as gene delivery system, the time point of expressionof the therapeutic gene into the host genome can be controlled bycontrolling the time point of retrotransposition of the L1-based genedelivery vector.

INDUSTRIAL APPLICABILITY

The herein disclosed nucleic acids, vectors and methods have industrialapplications.

-   -   1. The present invention facilitates random insertional        mutagenesis in cells and transgenic organisms in defined tissues        and during well defined and controlled periods of time. The        tightly controlled activation and deactivation of L1        retrotransposition at defined time points facilitates the        identification of gene functions in general and at defined        developmental stages. Conditional activation of the L1        retrotransposition in defined somatic tissues will help to        identify genes involved in cancer development. The relevance for        the pharmaceutical industry is obvious.    -   2. Ex vivo gene therapy approaches with        L1/Adenovirus-Hybridvectors or with other L1-based gene delivery        systems: If the therapeutic gene is part of the inducible        controlling the time point of L1 retrotransposition will define        the start of therapeutic gene expression. In order to be        introduced into the cell, the inducible L1-cassette of the        therapeutic gene has to be combined with an infectious virus.

The invention can be used to identify the effects of host-encoded orother proteins or components on L1 retrotransposition activity (whichcauses mutations that can lead to genetic disorders or cancer) in tissueculture cells. L1 transcription will be upregulated for the period oftime the host gene of interest is expressed in the cell. This way it isguaranteed that L1 expression and L1 transcription occurs only in thepresence of the gene product of interest.

EXAMPLES Example 1 General Experimental Approach

Detailed Experimental Protocol:

M2-HeLa cells (Hampf & Gossen, 2006, Anal Biochem.; 356(1):94-9) wereused together with the inducible L1-retrotransposition-reporter-plasmidptetO7CMV/L1.3blas (FIG. 2A). M2-HeLa cells express thetransactivator-protein rtTA. The cells were cultivated in D-MEM(BIOCHROM AG) with 4.5 g glucose (SIGMA), 10% FCS (BIOWEST), 2 mMglutamine (BIOCHROM AG), and 250 μg geneticine/ml (GIBCO or INVITROGEN)at 37° C. and 5% CO₂. 2×10⁵ M2-HeLa cells were seeded per well of asix-well plate. 24 hours later the cells were incubated with thetransfection reagent GeneJuice (Novagen) containing 1 μgptetO7CMV/L1.3blas (16976 bp) according to the instructions of themanufacturer. Concurrently, doxycycline (Dox, final concentration 100ng/ml) (SIGMA) were added to the medium. 24 hours after the addition ofptetO7CMV/L1.3blas the selection of transfected cells was initiated byadding hygromycin (final concentration, 300 μg/ml) (INVITROGEN) to themedium. Control samples contained neither hygromycin nor doxycycline.Every 48 hours the medium was replaced by fresh medium containing theabove cited concentrations of doxycycline and hygromycin. Three and fivedays after transfection of the vector construct, selection withhygromycin and the addition of doxycycline were terminated and aselection for retrotransposition events was started by addingblasticidine at a final concentration of 3 μg/ml (FIG. 2C). Transfectedcells that were grown in the absence of Dox resulted in only sporadicblasticidine resistant colonies (FIG. 2C, wells 1-3 and 7-9) indicatingthat L1 was barely expressed and only sporadic L1 retrotranspositionevents took place. Strikingly, adding dox to the cells for three and 5days led to a massive activation of L1-retrotransposition, which isreflected by ˜250 (FIG. 2C, well # 4-6) and ˜550 (FIG. 2B, well #10-12)blasticidine-resistant HeLa colonies, respectively. The experimentdemonstrates ‘proof-of-principle’ of temporal control ofL1-retrotransposition by adding Dox to the medium.

Example 2

The system described in example 1 is used to analyse the role ofdouble-strand break (DSB) repair enzymes during L1-retrotransposition.Dominant negative mutants, shRNA constructs, and siRNA acting on saidenzymes or on the expression of said enzymes are used to influence theactivity/expression of these enzymes. The Tet-On/Off system can be usedto activate L1-retrotransposition only during the time period the DSBrepair enzymes are influenced in the above described way. In this way,only those L1 retrotransposition events are analyzed that took place inthe presence of the modified DSB repair protein expression.

Example 3 Timecourse Experiment Demonstrating Induction of L1 ExpressionOver Time after Dox-Addition

Four T75-flasks containing 6×10⁶ M2-HeLa cells in DMEM-Medium each weretransfected with 6 μg of the inducible L1 reporter plasmidptet07CMV/L1.3blas (FIG. 3A).

One day after transfection, transfected M2-HeLa cells were selected forthe presence of the inducible L1 reporter construct by adding hygromycin(final cone. 300 μg/ml) to the growth medium.

DMEM medium containing hygromycin was changed twice a week.

After 17 days of hygromycin selection, ptet07CMV/L1.3blas-containingcells were harvested and cells were dispersed on eight 10-cm dishes with1.7×10⁶ cells per dish.

With the exception of two plates, doxycyclin was added to each plate toa final concentration of 200 ng/ml to induce transcription of theL1-reporter element localized on ptet07CMV/L1.3blas.

Cells were harvested 0, 6, 12 or 24 hours after dox-addition. For eachtimepoint, 3.4×10⁶ cells (two plates) were harvested and total RNA wasextracted (FIG. 3B) using the ‘Single-Step RNA Isolation Method’described recently (Chomzynski & Sacchi, 1987. Anal. Biochem. 162:156-9).

For each time point before and after dox-induction, 15 μg of denaturedtotal RNA were subjected to electrophoresis in a horizontal 1.2% agarosegel containing morpholinepropane sulfonic acid (MOPS) buffer and 6%formaldehyde (Sambrook et al. 1989, Molecular Cloning: a laboratorymanual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.).

Subsequently, the RNA was transferred to a nylon membrane by theNorthern blot procedure (FIG. 3B).

The probe used to detect inducible L1 transcripts was a 674-bpPvuII-EcoRI fragment covering the 5′-half of the blasticidin-gene(Blast) in the reporter cassette (FIG. 3A). Transcription of theconstitutively expressed actin gene was detected with the help of a643-bp fragment of the actin cDNA generated by PCR with oligonucleotidesSW38 (5′-CACCCACAACGTGCCCATTTATGAG-3′) and SW39(5′-TITGCGGTGGACGATGGAAGG-3′). The DNA probes were labelled forhybridization with (α32P)dATP by random priming (Feinberg & Vogelstein,1983, Anal. Biochem. 132: 6-13).

Result: Induction of L1-blast transcription occurred between 6 and 12hours after Dox-addition (FIG. 3B). L1-blast expression is slightlyreduced after 24 hours due to the instability of Doxycyclin (Barry andBadal, 1978, Current Microbiology, 1:33-36) and because it is consumedby the cells over time.

1. A nucleic acid molecule comprising: an autonomous non-long terminalrepeat (LTR) retrotransposon that lacks an active endogenous promoter;and an operably linked inducible first promoter exogenous to theretrotransposon.
 2. The nucleic acid molecule of claim 1, wherein thefirst promoter comprises a tetracycline-response element (TRE) operablylinked to a TRE-responsive promoter.
 3. The nucleic acid molecule ofclaim 2, wherein the TRE-responsive promoter is a cytomegaloviruspromoter.
 4. The nucleic acid molecule of claim 1, wherein theretrotransposon comprises a heterologous gene selected from the groupconsisting of a reporter gene, a therapeutic gene, and a selectablemarker gene.
 5. The nucleic acid molecule of claim 4, wherein thereporter gene is selected from the group consisting of a greenfluorescent protein (GFP) gene, an enhanced-GFP (EGFP) gene, and abeta-galactose gene.
 6. The nucleic acid molecule of claim 4, whereinthe selectable marker gene confers resistance to an antibiotic.
 7. Thenucleic acid molecule of claim 6, wherein the antibiotic is selectedfrom the group consisting of blasticidin and neomycin.
 8. The nucleicacid molecule of claim 4, wherein the heterologous gene is operablylinked to a second promoter.
 9. The nucleic acid molecule of claim 8,wherein the first and second promoters are not the same promoter. 10.The nucleic acid molecule of claim 8, wherein at least one of the firstand second promoters is selected from the group consisting of atissue-specific promoter and a cell-type specific promoter.
 11. Thenucleic acid molecule of claim 8, wherein the second promoter is aninducible promoter.
 12. The nucleic acid molecule of claim 1, whereinthe retrotransposon comprises a mammalian retrotransposon ORF1, ORF2, 3′UTR, and poly A signal.
 13. The nucleic acid molecule of claim 12,wherein the mammal is selected from the group consisting of a human, arat, and a mouse.
 14. The nucleic acid of claim 12, wherein theretrotransposon is a mammalian long interspersed element 1 (LINE-1) typeretrotransposon.
 15. The nucleic acid molecule of claim 14, wherein theLINE-1 is codon-optimized.
 16. The nucleic acid molecule of claim 12,comprising a heterologous gene in the 3′ UTR, the heterologous genebeing selected from the group consisting of a reporter gene, atherapeutic gene, and a first selectable marker gene.
 17. The nucleicacid molecule of claim 16, wherein the reporter gene is selected fromthe group consisting of a green fluorescent protein (GFP) gene, anenhanced-GFP (EGFP) gene, and a beta-galactose gene.
 18. The nucleicacid molecule of claim 16, wherein the selectable marker gene confersresistance to an antibiotic.
 19. The nucleic acid molecule of claim 16wherein the heterologous gene in the 3′ UTR can be transcribed onlyafter retrotransposition of the retrotransposon.
 20. The nucleic acidmolecule of claim 16, wherein the heterologous gene is operably linkedto a second promoter.
 21. The nucleic acid molecule of claim 20, whereinthe first and second promoters are not the same promoter.
 22. Thenucleic acid molecule of claim 20, wherein at least one of the first andsecond promoters is selected from the group consisting of atissue-specific promoter and a cell-type specific promoter.
 23. Thenucleic acid molecule of claim 1, wherein the molecule is a vectorconstruct selected from the group consisting of a viral vector and aplasmid.
 24. The nucleic acid molecule of claim 20, wherein theheterologous gene is positioned in the retrotransposon 3′ UTR such thatit can be transcribed only after retrotransposition of theretrotransposon into a cell genome and wherein the molecule furthercomprises, outside of the retrotransposon, a second selectable markergene.
 25. A method for transfecting mammalian cells comprising the stepsof: (a) transfecting into a mammalian cell that expresses a reversetetracycline-controlled transactivator-protein (rtTA) a nucleic acidmolecule that comprises: an autonomous non-long terminal repeat (LTR)retrotransposon having a mammalian retrotransposon ORF1, ORF2, 3′ UTRand poly A signal but lacking an active endogenous promoter, aninducible first promoter exogenous to and operably linked to theretrotransposon, a first selectable marker gene exogenous to theretrotransposon, and a second selectable marker gene provided in the 3′UTR and operably linked to a second promoter; (b) exposing thetransfected cells to doxycycline and a first selection agent thatpositively selects cells expressing the first selectable marker, for atime sufficient to select the cells expressing the first selectablemarker; (c) exposing the selected cells to a second selection agent thatpositively selects cells expressing the second selectable marker, for atime sufficient to select the cells expressing the second selectablemarker; and (d) recovering the cells selected in step (c).
 26. Themethod of claim 25, wherein the mammalian cells are of human origin.